DKK1 (SEQ ID NO:1) encodes a secreted protein which plays a crucial role in head formation in vertebrate development, and is known as a negative regulator of the Wnt-signaling pathway in colon cancer cells (Niida A. et al. Oncogene 2004; 23:8520-6, Gonzalez-Sancho J M. et al. Oncogene 2005; 24:1098-103). The over-expression of DKK1 was previously reported as periodically arising in hepatoblastomas, Wilms' tumors, hepatocellular carcinomas (HCC), prostate cancer, and breast cancer, indicating a potential oncogenic function of DKK1 (Wirths O. et al. Lab Invest 2003; 83:429-34, Patil M A. et al. Oncogene 2005; 24:3737-47, Hall C L. et al. Cancer Res. 2005; 65:7554-60, Forget M A. et al. Br J Cancer. 2007; 96:646-53). In addition, serum concentrations of DKK1 protein have been shown to be increased in patients with multiple myeloma (Politou M C. et al. Int J Cancer. 2006; 119:1728-31). Furthermore, inhibition of DKK1 using an anti-DKK1 antibody has been shown to reverse the bone-destructive rheumatoid arthritis in mice to the bone-forming osteoarthritis (Diarra D et al. Nat. Med. 2007; 13:156-63). More recently, Yaccoby et al. demonstrated that antibody-based inhibition of DKK1 could suppress tumor-induced bone resorption and multiple myeloma growth in vivo. Specifically, daily subcutaneous injection of a neutralizing DKK1 antibody in the area surrounding myelomatous bone was shown to ameliorate bone turnover, presumably by increasing osteoblastogenesis and reducing osteoclastgenesis (Blood. 2007; 109:2106-11).
The role of DKK1 in promoting the development of bone lesions has also been studied in prostate cancer. PC-3, the osteolytic cancer cell line, was shown to revert to an osteoblastic phenotype when transfected with shRNA targeting DKK1. In addition, transfection of DKK1 into the osteoblastic prostate cancer cell line C4-2B, which normally induces a mix of osteoblastic and osteolytic lesions, caused the cells to develop osteolytic tumors in SCID mice (Hall C L. et al. Cancer Res. 2005; 65:7554-60).
The present inventors have screened for therapeutic target molecules using the following strategy: (I) Identifying up-regulated genes in lung cancer by genome-wide cDNA microarray system (Kikuchi T et al. Oncogene. 2003; 22:2192-205, Kikuchi T et al. Int J Oncol 2006; 28:799-805, Kakiuchi S et al. Mol Cancer Res 2003; 1:485-99, Kakiuchi S et al. Hum Mol. Genet. 2004; 13:3029-43, Taniwaki M et al. Int J Oncol 2006; 29:567-75, Yamabuki T et al. Int J Oncol: 2006; 28: 1375-84), (II) Verifying the candidate genes for its no or very low level of expression in normal tissues by northern-blotting (Saito-Hisaminato A et al. DNA Res. 2002; 9:35-45, Ochi K et al. J Hum Genet. 2003; 48:177-82), (III) Validating the biological significance of over-expression using tissue microarrays containing hundreds of archived lung-cancer samples and RNAi assay (Kato T et al. Cancer Res. 2005; 65:5638-46, Furukawa C. et al. Cancer Res. 2005; 65:7102-10, Ishikawa N et al. Cancer Res. 2005; 65:9176-84, Suzuki C et al. Cancer Res. 2005: 65:11314-11325, Ishikawa N et al. Cancer Sci. 2006; 97:737-745, Takahashi K et al. Cancer Res. 2006; 66:9408-9419), (IV) Evaluating usefulness as a serum diagnostic/prognostic biomarker for lung cancer by ELISA (Ishikawa N et al. Cancer Res. 2005; 65:9176-84, Ishikawa N et al. Clin Cancer Res. 2004; 10:8363-70. Yamabuki T et al. Cancer Res 2007; 67:2517-2525), if they are tumor-specific trans-membrane or secretory proteins.
Using this approach, the inventors recently identified Dickkopf-I (DKK1) as a novel serological and histochemical biomarker as well as a therapeutic target for lung and esophageal cancers (WO2007/01367 1, Yamabuki T. et al. Cancer Res 2007; 67:2517-2525, the contents of which are incorporated by reference). A high level of DKK1 expression was associated with poor prognosis of patients with non-small cell lung cancer (NSCLC) as well as esophageal squamous cell carcinoma (ESCC). In addition, present inventors identified that exogenous expression of DKK1 increased the invasive activity of mammalian cells, suggesting that DKK1 may play a significant role in progression of human cancer. An ELISA system was established to measure serum levels of DKK1 and found that serum DKK1 levels were significantly higher in lung and esophageal cancer patients than in healthy controls. From these findings, the inventors then focused on DKK1 as a potential target for the generation of therapeutic antibodies applicable to cancer patients.